In conventional fluorescence microscopy, more than 90% of the observed fluorescence may originate from parts of the sample that are out of focus and this scattered light from planes above and below the area-of-interest obscures the detail of the specimen within the desired plane of focus.
Confocal microscopes overcome this problem by using a stationary pinhole located in a conjugate image plane which serves to reject light that originates outside the plane of focus and as such, this improves the apparent spatial resolution of the image.
As elegant a solution as this may be, taking the example of a confocal laser scanning microscope (CLSM), sequential scanning on a point by point basis limits the speed of image acquisition; as a result even the fastest commercial instruments are unable to resolve the temporal dynamics of the fastest cellular events.
An alternative approach to faster scanning that suffers neither from reduced image quality nor reduced optical sectioning is to scan multiple beams in parallel and use an array of pinholes and detectors to collect the image. Scanning disk systems such as the Nipkow disk are based on a spinning mask of pinholes that can simultaneously illuminate many discrete points. When the disk spins at high speed, laser light passes through the pinholes and illuminates the whole specimen almost simultaneously. A camera is used to collect the light that passes back through the pinholes. Optical sectioning occurs because light emitted above or below the focal plane does not return through the pin holes.
However, a major disadvantage of scanning disk systems is that they generally suffer from very low light throughput. Much of the illumination light is rejected by the disc because the spacing between neighbouring pinholes must be large to maintain the confocal effect (light that would otherwise be rejected may be able to pass through a neighbouring pinhole instead). In an ideal case, image acquisition speed should be limited by the camera frame rate but in practice, the actual speed is limited by the low amount of light that passes through the pinholes and the sensitivity of, for example, CCD cameras, necessitating longer exposure times.
The introduction of microlenses to spinning Nipkow disks, that enhance the transmission of excitation light, and electron magnifying (EM) CCD cameras, which improve detection efficiency, have improved the utility of these systems but the quality of results obtained from these systems depends entirely on the brightness of the specimen and so their efficacy depends on the particular application. However, low light throughput remains an issue. The Applicant has identified that part of the problem lies in being able to make use of as much light as possible. Nipkow disks have other significant limitations. First, they have fixed pinhole diameters and spacing. These two factors determine the level of optical sectioning and they can only be optimised for one particular numerical aperture of the objective lens. Although some systems may allow the disk to be changed, this would be inconvenient and require re-alignment of the optical pathway.
This problem has been overcome to an extent with the development of optically sectioning microscopes that utilise a digital micromirror device (DMD) as a spatial light modulator to provide optical sectioning (inc. fluorescence, widefield and confocal) at high spatial resolution and improved frame rates (in certain examples up to 100 Hz). The DMD acts as a solid state Nipkow disk but with the added ability to change the “pinhole” size and separation and to control the light intensity on a mirror by mirror basis. Different pinhole patterns can also be used over different regions of the field of view to allow alterations in light intensity or differing levels of optical sectioning within a field of view. The DMD can also be used for spatially defined photo-activation and so the system can be used for photolytic release of caged compounds, photoactivation or photoconversion of fluorescent proteins and fluorescent recovery after photobleaching (FRAP) measurements.
Nonetheless, it is an object of the present invention to provide an improved optical arrangement that overcomes one or more deficiencies of existing confocal microscopes.
However, it is realised that the present invention has utility in broader applications than solely confocal microscopes.
Therefore, it is a further object of the present invention to provide an improved optical arrangement that may result in improved performance in optical imaging systems and the like.